Sc 7392 hrp

Western blotting on IP samples was done by running a 10% SDS–PAGE gel, and the proteins were transferred on a 0.45 μm nitrocellulose membrane (10600002 Amersham, GE Healthcare). 1% of Input and 20% of IP fraction were used for the samples to be run on the gel. The membrane was blocked with 5% BSA in PBS for 1 h at room temperature. The membrane was then incubated overnight at 4°C with anti‐HA (sc805, Santa Cruz, RRID: AB_631618) at a dilution of 1:500. A goat anti‐rabbit HRP conjugate (sc2004, Santa Cruz, RRID: AB_631746) in a dilution of 1:5,000 was used after washing the membrane with PBS/0.1% Tween‐20 for 10 min (three times). For PTIWI01‐3XFLAGHA IP, the membrane was incubated with either anti‐HA (sc‐7,392 HRP, Santa Cruz, RRID: AB_627809) in a dilution of 1:500 or with anti‐GFP (ab290, Abcam, RRID: AB_303395) in a dilution of 1:1,000. The secondary antibody incubation was done for 1 h at room temperature, and the membrane was washed thrice with PBS/0.1% Tween‐20 for 10 min. The membrane was then washed once for 5 min with 1× PBS before imaging. The membrane was scanned using chemiluminescence settings on an Amersham Imager 600 (GE Healthcare).

Monkey COS-7 cells, human HEK293 and U2OS were grown in DMEM medium with 4,500 mg l⁻¹ glucose supplemented with 10% bovine calf serum (Hyclone). HeLa cells were grown in Eagle's MEM medium supplemented with L-glutamine, sodium bicarbonate, sodium pyruvate and 10% bovine calf serum. Transient transfections were performed with Lipofectamine 2000 according to recommended protocols. Typically, a total of 5 μg plasmid DNA was used per 60-mm dish; lysates were harvested 18 h later in ice-cold lysis buffer (0.5 ml; 25 mM HEPES, pH 7.3, 100 mM KCl, 5 mM MgCl₂, 20 mM β-glycerophosphate, 5% glycerol, 0.5% Triton X-100, 5 mM dithiothreitol, 0.5 mM phenylmethyl sulfonyl fluoride, 1 mM Na₃VO₄ and 1 × protease inhibitor cocktail (Roche)). To test co-immunoprecipitation of proteins, the lysates were clarified by centrifugation (14,000g) and the clarified lysates were incubated while rolling (2 h) with 20 μl M2 anti-Flag Sepharose (Sigma-Aldrich, A2220). Rabbit anti-Flag (Sigma-Aldrich, F7425) or horseradish peroxidase-coupled anti-HA (Santa Cruz Biotechnology, sc-7392 HRP, 1 μg ml⁻¹) were used for western blot analysis.